ABSTRACT Trinucleotide repeat expansions are a common cause of several neurodegenerative diseases including Huntington?s disease (HD). HD is caused by expansion of CAG repeats in the first exon of huntingtin (HTT) that is translated as a polyglutamine tract. The protein aggregates formed from the polyglutamine-containing peptide are the main cause of neuronal cell death, although recent results have suggested that the RNA repeats itself may also be directly responsible for neurotoxicity. Therapeutic strategies directly targeting mutated HTT mRNA, such as antisense oligonucleotides (ASO), have produced promising results. However, difficulties in ASO delivery, the unknown effects of ASO on structured CAG repeats, and possibility that ASOs may disrupt both expanded and normal transcripts remain unresolved. In this proposal, we propose to use our Artificial SiteSpecific RNA Endonucleases (ASREs) technology to design CAG repeat specific RNA endonuclease to destroy expanded pathogenic HTT RNAs. ASREs contain RNA binding domains isolated from PUF proteins, which consist of a series of ~36 amino acid modules that recognize one specific ribonucleotide. In a proof of concept study, we designed ASREs against expanded CAG repeats, which are present in the DMPK gene, which is associated with the (CUG)n repeats disorder, Myotonic Dystrophy type 1 (DM1). We demonstrated that (CUG)n specific ASREs specifically degrade pathogenic DMPK mRNAs with minimal effect on wild-type alleles in cells derived from DM1 patients and corrected several molecular markers of DM1 disese. We seek to develop a protein-based therapeutic approach for HD by designing ASREs that recognize CAG repeats in mutated HTT mRNA. In the long term, combined with gene delivery vectors, ASREs may provide a new route for targeted therapy. To assess the feasibility of this approach we will first engineer ASREs that specifically recognizes 8-nt or 10-nt RNA sequences in three different frames of the (CAG)n repeat (CAGCAGCAGC, AGCAGCAGCA, GCAGCAGCAG) and use a yeast three hybrid system to identify the PUF domain that has highest affinity for (CAG)25 repeats with low affinity for (CAG)5 repeats. PUF domains that selectively recognizes (CAG)25 at efficiencies 5-fold higher than (CAG)5 repeats will be cloned into a piggyBac cumate-inducible transposon vector to create a (CAG)n specific ASRE for cell studies [PB-ASRE(CAG)n]. Second, we will transduce PB-ASRE(CAG)n into heterozygous knockin embryonic stem (ES) cells that express endogenous levels of either human exon 1-containing normal (Htt20Q) or mutant (Htt140Qt) alleles together with a normal mouse 7Q allele (Htt7Q). Undifferentiated ES Cells and ES cell derived neurons will be cultured plus or minus cumate to quantitate the level of knockdown of the expanded mRNA relative to the normal allele by qPCR and quantitate the level of knockdown of the polyglutamine-containing peptide relative to the normal peptide by Western blotting. We seek to demonstrate that induction of PB-ASRE(CAG)n results in preferential downregulation of mutant RNA and protein (at least three-fold preferential knockdown of the mutant RNA and protein). Once feasibility is demonstrated, Phase 2 studies will focus on the development of research grade adenoviral associated vectors (AAV) that constitutively express ASRE(CAG)n to develop gene delivery protocols and for initial efficacy and safety studies in animal models of HD before progressing to production of clinical grade AAV ASRE therapeutics for IND enabling safety and efficacy studies.